Gill:Preparing phage specimens for TEM

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The following are two methods of preparing a phage sample for viewing using a transmission electron microscope. In both protocols, the phages adhere to a thin layer of carbon film that is mounted on a copper grid and are negatively stained using heavy metal atoms (uranium or tungsten). The classic method makes use of grids that have already been coated with a layer of carbon and formvar. The classic method is simpler, but it can be more cost-effective to prepare the grids yourself using the Valentine method.
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=Classic method=
=Classic method=
==Materials==
==Materials==
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*5 mM MgSO4 (or lambda dil)
*5 mM MgSO4 (or lambda dil)
*2% Uranyl acetate in water (or 2% phosphotungstic acid) <br>''Uranyl acetate is highly toxic—wear gloves and safety glasses while handling. Protect from light. If a cloudy precipitate forms, discard the stain. The stain should last for six months to a year.''
*2% Uranyl acetate in water (or 2% phosphotungstic acid) <br>''Uranyl acetate is highly toxic—wear gloves and safety glasses while handling. Protect from light. If a cloudy precipitate forms, discard the stain. The stain should last for six months to a year.''
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*300 mesh carbon grids coated with formvar carbon film
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*300 mesh copper grids coated with formvar carbon film
*Filter paper cut into small, triangular pieces
*Filter paper cut into small, triangular pieces
*Petri dish with a grid mat
*Petri dish with a grid mat
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==Procedure==
==Procedure==
# If the plate lysate was prepared in a nutrient rich media such as LB…
# If the plate lysate was prepared in a nutrient rich media such as LB…
-
## Centrifuge 100 uL of the lysate at 14K RPM for two hours at room temperature.
+
## Centrifuge 1 ml of the lysate at 14K RPM for two hours at room temperature.
-
## Discard the supernatant and pipette 20 uL 5 mM MgSO4 onto the pellet.
+
## Discard the supernatant and pipette 200 uL 5 mM MgSO4 onto the pellet.
## Allow the pellet to soak overnight in a 5°C fridge.
## Allow the pellet to soak overnight in a 5°C fridge.
## Re-suspend the pellet by gently pipetting up and down. Do not vortex.
## Re-suspend the pellet by gently pipetting up and down. Do not vortex.
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==Procedure==
==Procedure==
# If the plate lysate was prepared in a nutrient rich media such as LB…
# If the plate lysate was prepared in a nutrient rich media such as LB…
-
## Centrifuge 100 uL of the lysate at 14K RPM for two hours at room temperature.
+
## Centrifuge 1 ml of the lysate at 14K RPM for two hours at room temperature.
-
## Discard the supernatant and pipette 20 uL 5 mM MgSO4 onto the pellet.
+
## Discard the supernatant and pipette 200 uL 5 mM MgSO4 onto the pellet.
## Allow the pellet to soak overnight in a 5°C fridge.
## Allow the pellet to soak overnight in a 5°C fridge.
## Re-suspend the pellet by gently pipetting up and down. Do not vortex.
## Re-suspend the pellet by gently pipetting up and down. Do not vortex.
-
# Dilute the phage sample 2x and 5x in 5 mM MgSO4.
+
# Dilute the phage sample 2x and 4x in 5 mM MgSO4.
# Shake some copper grids into a glass culture tube. Pour enough isopropanol into the tube to cover the grids.
# Shake some copper grids into a glass culture tube. Pour enough isopropanol into the tube to cover the grids.
# Vortex the grids. <br>''Optional: let the grids soak in isopropanol for a few hours.''
# Vortex the grids. <br>''Optional: let the grids soak in isopropanol for a few hours.''
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# Put a funnel in a beaker. Fold the filter paper into a cone and place in the funnel.
# Put a funnel in a beaker. Fold the filter paper into a cone and place in the funnel.
# Vortex the grids and quickly pour into the funnel so the grids do not stick to the sides of the tube. Pour off the isopropanol or let it drain into the beaker.
# Vortex the grids and quickly pour into the funnel so the grids do not stick to the sides of the tube. Pour off the isopropanol or let it drain into the beaker.
-
# Take the filter paper out of funnel and touch it to the filter paper in the petri dish to transfer the grids. Use forceps to transfer any remaining grids.
+
# Take the filter paper out of the funnel and touch it to the filter paper in the petri dish to transfer the grids. Use forceps to transfer any remaining grids.
# Allow the grids to dry with the lid off for about an hour.
# Allow the grids to dry with the lid off for about an hour.
# Place a round grid mat into a petri dish.
# Place a round grid mat into a petri dish.
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# With the carbon side facing up, insert the carbon film into the drop of phage sample at a shallow (~30 degree) angle. Hold on to an edge of the mica with the forceps (don’t fully submerge the carbon film, so that you can pull it back out). Wait for 1 minute. The carbon should detach and float on top of the drop, adhering phage particles on its bottom side.
# With the carbon side facing up, insert the carbon film into the drop of phage sample at a shallow (~30 degree) angle. Hold on to an edge of the mica with the forceps (don’t fully submerge the carbon film, so that you can pull it back out). Wait for 1 minute. The carbon should detach and float on top of the drop, adhering phage particles on its bottom side.
# Pull the film out of the sample and place it on a drop of stain. Leave the film in the stain for 10-15 seconds.
# Pull the film out of the sample and place it on a drop of stain. Leave the film in the stain for 10-15 seconds.
-
# Pick up a grid using forceps and touch it to the carbon floating on the surface of the stain. The carbon film should stick to the grid.<br> ''Use either the carbon side or the shiny side of the grid—just be consistent.''
+
# Pick up a grid using forceps and touch it to the carbon floating on the surface of the stain. The carbon film should stick to the grid.<br> ''Use either the dull or the shiny side of the grid—just be consistent.''
# Pick up the grid and touch a triangular piece of filter paper to the edges to wick off the stain.
# Pick up the grid and touch a triangular piece of filter paper to the edges to wick off the stain.
# Place the grid onto a grid mat to dry, sample side up. Note which grids are in which sectors. Store the grids in a desiccator.
# Place the grid onto a grid mat to dry, sample side up. Note which grids are in which sectors. Store the grids in a desiccator.

Current revision

The following are two methods of preparing a phage sample for viewing using a transmission electron microscope. In both protocols, the phages adhere to a thin layer of carbon film that is mounted on a copper grid and are negatively stained using heavy metal atoms (uranium or tungsten). The classic method makes use of grids that have already been coated with a layer of carbon and formvar. The classic method is simpler, but it can be more cost-effective to prepare the grids yourself using the Valentine method.

Contents

Classic method

Materials

  • Phage sample
  • 5 mM MgSO4 (or lambda dil)
  • 2% Uranyl acetate in water (or 2% phosphotungstic acid)
    Uranyl acetate is highly toxic—wear gloves and safety glasses while handling. Protect from light. If a cloudy precipitate forms, discard the stain. The stain should last for six months to a year.
  • 300 mesh copper grids coated with formvar carbon film
  • Filter paper cut into small, triangular pieces
  • Petri dish with a grid mat
  • Parafilm
  • Forceps

Procedure

  1. If the plate lysate was prepared in a nutrient rich media such as LB…
    1. Centrifuge 1 ml of the lysate at 14K RPM for two hours at room temperature.
    2. Discard the supernatant and pipette 200 uL 5 mM MgSO4 onto the pellet.
    3. Allow the pellet to soak overnight in a 5°C fridge.
    4. Re-suspend the pellet by gently pipetting up and down. Do not vortex.
  2. Place a paper towel on the bench. Cut a strip of Parafilm, remove the paper backing, and place it on the paper towel.
  3. Pipette 30 ul of 5 mM MgSO4 onto the Parafilm and add 10 uL of the phage sample to the drop. Gently pipette up and down to mix.
  4. Pipette 30 ul of 2% uranyl acetate onto the Parafilm for each of the grids to be prepared.
  5. Pick up a grid using forceps and place it on a drop of phage sample, carbon side down. Wait for 1 minute.
  6. Pick up the grid and place it on the drop of stain. Leave the grid on the stain for 10-15 seconds (or 1 minute if using PTA).
  7. Pick up the grid and touch a triangular piece of filter paper to the edges to wick off the stain.
  8. Place the grid onto a grid mat to dry, carbon side up. Note which grids are in which sectors. Store the grids in a desiccator.

Valentine method

Materials

  • Phage sample
  • 5 mM MgSO4 (or lambda dil)
  • 2% Uranyl acetate in water
    Uranyl acetate is highly toxic—wear gloves and safety glasses while handling. Protect from light. If a cloudy precipitate forms, discard the stain. The stain should last for six months to a year.
  • Isopropanol
  • Uncoated 300 mesh copper grids
  • Carbon film (mica coated with carbon to a thickness of 10-15 nm)
  • Whatman filter paper, grade 1
  • Forceps
    Make sure the tips of the forceps are not blunt or damaged.
  • Small scissors
  • Grid mats
  • Glass culture tube
  • Funnel
  • Beaker
  • Petri dishes
  • Parafilm
  • Scotch tape

Procedure

  1. If the plate lysate was prepared in a nutrient rich media such as LB…
    1. Centrifuge 1 ml of the lysate at 14K RPM for two hours at room temperature.
    2. Discard the supernatant and pipette 200 uL 5 mM MgSO4 onto the pellet.
    3. Allow the pellet to soak overnight in a 5°C fridge.
    4. Re-suspend the pellet by gently pipetting up and down. Do not vortex.
  2. Dilute the phage sample 2x and 4x in 5 mM MgSO4.
  3. Shake some copper grids into a glass culture tube. Pour enough isopropanol into the tube to cover the grids.
  4. Vortex the grids.
    Optional: let the grids soak in isopropanol for a few hours.
  5. Place a couple layers of filter paper into a petri dish.
  6. Put a funnel in a beaker. Fold the filter paper into a cone and place in the funnel.
  7. Vortex the grids and quickly pour into the funnel so the grids do not stick to the sides of the tube. Pour off the isopropanol or let it drain into the beaker.
  8. Take the filter paper out of the funnel and touch it to the filter paper in the petri dish to transfer the grids. Use forceps to transfer any remaining grids.
  9. Allow the grids to dry with the lid off for about an hour.
  10. Place a round grid mat into a petri dish.
  11. Place a layer of filter paper into another petri dish. Tape one side of a sheet of carbon film to the filter paper.
  12. Cut filter paper into small, triangular pieces.
  13. Place a paper towel on the bench. Cut a strip of Parafilm, remove the paper backing, and place it on the paper towel.
  14. Cut off the bottom edge of the carbon film and discard.
  15. Mix the diluted phage samples by flicking. Pipette 50 ul of each dilution onto the Parafilm.
  16. Pipette 50 ul of 2% uranyl acetate onto the Parafilm for each of the grids to be prepared.
    The sample drops can be re-used to make multiple grids, but the drops of stain can only be used once.
  17. Wipe off the tips of the forceps on the paper towel.
  18. Holding the carbon film with forceps, cut off a 5 mm strip.
  19. With the carbon side facing up, insert the carbon film into the drop of phage sample at a shallow (~30 degree) angle. Hold on to an edge of the mica with the forceps (don’t fully submerge the carbon film, so that you can pull it back out). Wait for 1 minute. The carbon should detach and float on top of the drop, adhering phage particles on its bottom side.
  20. Pull the film out of the sample and place it on a drop of stain. Leave the film in the stain for 10-15 seconds.
  21. Pick up a grid using forceps and touch it to the carbon floating on the surface of the stain. The carbon film should stick to the grid.
    Use either the dull or the shiny side of the grid—just be consistent.
  22. Pick up the grid and touch a triangular piece of filter paper to the edges to wick off the stain.
  23. Place the grid onto a grid mat to dry, sample side up. Note which grids are in which sectors. Store the grids in a desiccator.
  24. Image the grids at a magnification of 25K. Higher magnifications have less contrast and are more likely to burn a hole through the grid.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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